The Study On The Mechanism Of Glutamine Catabolism In Sepsis-associated Acute Kidney Injury

Objective:Sepsis is a life-threatening clinical syndrome.Severe sepsis is often accompanied by organ dysfunction.The kidney is the most commonly affected organ,that is,sepsis-associated acute kidney injury(SA-AKI).Glutamine is a nutrient with anti-inflammatory and immunomodulatory properties.Sepsis can lead to obvious disorder of glutamine flow among organs.The present study mainly discusses the role and molecular mechanism of glutamine metabolism in the development of SA-AKI.Methods:Firstly,lipopolysaccharide(LPS)was used to stimulate mouse renal tubular epithelial cells(TKPTS)to establish an in vitro SA-AKI model.The mouse model of SA-AKI was constructed via cecal ligation and puncture.Cell proliferation,lactate dehydrogenase content and ferroptosis-related indicators,including glutathione,malondialdehyde(GSH),reactive oxygen species and ferroptosis-inhibitory protein(xCT and GPx4)levels in TKPTS and mouse kidney,were detected by reagent kits,flow cytometry,immunofluorescence and Western blotting.Secondly,the expression of glutamine metabolism-related genes in the kidney of SA-AKI mice was analyzed by public data.Glutaminase(GLS),a key enzyme in the first step of glutamine metabolism,was detected in vivo and in vitro models.The effect of inhibiting glutamine metabolism and supplementing metabolic intermediates on the ferroptosis was detected in vitro.The expression of mammalian target of rapamycin(mTOR),activating transcription factor 4(ATF4)and nuclear factor erythroid 2-related factor 2(Nrf2)were detected via western blotting and Immunofluorescence.Rapamycin was used to inhibit mTOR activity in glutamine-deprived TKPTS.siRNAs were used to silence Atf4 or Nrf2.Ferroptosis-related indicators were detected and compared among different groups.Finally,the tubular-specific knockout of Gls were established in mice.SA-AKI was constructed via cecal ligation and puncture.The mortality,renal function and ferroptosis-related indicators were compared.Results:Compared with the control group,the model group showed ferroptosis,decreased glutathione,increased malondialdehyde and reactive oxygen species,and decreased expression of xCT and GPx4.Public data analysis showed that the expression of glutamine metabolism-related genes in SA-AKI mice was up-regulated.The expression of GLS in LPS-stimulated TKPTS and SA-AKI mouse kidneys increased.Inhibition of glutamine metabolism in vitro by drugs or glutamine deprivation reduced LPS-induced ferroptosis in TKPTS,while supplementation of glutamine metabolic intermediates in the deprivation group reversed the effect on ferroptosis.After LPS stimulation,mTOR was activated in the deprivation group,and the expression of ATF4 and Nrf2 was up-regulated and transferred to the nucleus.After inhibiting mTOR in the deprivation group by rapamycin,compared with the LPS group without glutamine,the expression of ATF4 and Nrf2 was down-regulated,and ferroptosis increased.When siRNA silenced Atf4 in glutamine-deprived LPS group,mTOR was activated and Nrf2 expression was down-regulated,and ferroptosis was increased.When Nrf2 was silenced in glutamine-deprived LPS group by siRNA,mTOR was activated,no difference was observed on ATF4 expression,and ferroptosis increased.Finally,we successfully constructed renal tubular specific knockout Gls mice.Compared with the SA-AKI mice without gene knockout,renal ferroptosis reduced in the knockout group,accompanied by improved renal function and decreased mortality.Conclusions:In SA-AKI,glutamine catabolism is enhanced in renal tubule.Inhibition of glutamine metabolism activates the mTOR pathway,and mTOR/ATF4 stabilizes the xCT-GSH-GPx4 antioxidant system in renal tubular cells by regulating Nrf2,thereby reducing ferroptosis in renal tubular cells.Specific knockout of Gls in renal tubule can improve renal function and survival in SA-AKI mice,suggesting that renal glutamine metabolism is involved in the progression of SA-AKI by regulating ferroptosis.Part Ⅰ:Ferroptosis in sepsis associated acute kidney injuryObjective:To investigate whether ferroptosis occurs in renal tubular cells in the context of sepsis-associated acute kidney injury(SA-AKI).Methods:The renal tubular epithelial cells(TKPTS)were stimulated by lipopolysaccharide(LPS)in vitro,and the SA-AKI mouse model was established via cecal ligation and puncture in vivo.Cell proliferation,lactate dehydrogenase content,and ferroptosis-related indicators,including GSH,malondialdehyde,reactive oxygen species,and ferroptosis-inhibiting protein(xCT and GPx4)levels were detected by reagent kits,flow cytometry,immunofluorescence,and Western blotting.The renal function and renal pathological changes were detected.The morphological changes of renal tubular mitochondria were detected by transmission electron microscopy.Results:In vitro,cell death was obvious at LPS concentration of 100μg/mL and stimulation time of 24 h.Therefore,this regimen was used in subsequent experiments.Compared with the control group,GSH decreased,malondialdehyde and reactive oxygen species increased,xCT and GPx4 expression decreased in LPS-stimulated TKPTS.In vivo,mice undergoing cecal ligation and perforation showed significant renal dysfunction 24 hours later,manifested by increased serum creatinine and urea nitrogen.Pathological staining showed vacuole-like changes,exfoliation and dilation of renal tubular epithelial cells.Besides,GSH decreased,malondialdehyde and reactive oxygen species increased,and GPx4 expression decreased in SA-AKI mice.Electron microscopy showed that the mitochondria of cortical renal tubules in SA-AKI mice were smaller,accompanied by edema and cristae disruption,suggesting ferroptosis.Conclusions:Ferroptosis occurred in SA-AKI.Part Ⅱ:Glutamine metabolism in the activation of ferroptosis in renal tubuleObjective:To investigate whether glutamine metabolism can regulate ferroptosis of renal tubular cells in SA-AKI.Methods:Public data were collected from the Gene Expression Omnibus(GEO)database to analyze the transcriptome expression of glutamine metabolism-related genes in SA-AKI.The expression of GLS in vivo and in vitro was detected by immunofluorescence and immunoblotting.In vitro,BPTES pretreatment or removal of glutamine in the medium were performed in order to inhibit glutamine metabolism.Ferroptosis-related indicators were detected.Then,the metabolite α-ketoglutarate(α-KG)was supplemented,and then stimulated in the glutamine-deprived group with LPS to detect ferroptosis.Results:In GEO database,the expression of genes related to renal glutamine metabolism was increased in SA-AKI mice.Immunofluorescence and western blotting indicated increased GLS expression in SA-AKI in vivo and in vitro.Compared with the LPS group,inhibiting glutamine metabolism resulted in decreased and lactate dehydrogenase activity,increased GSH,reduced malondialdehyde and reactive oxygen species,and increased expression of xCT and GPx4,that is,ferroptosis was inhibited.The effect was reversed when α-KG was supplemented in the glutamine-deprived group.Conclusions:Glutamine metabolism in renal tubules was enhanced in SA-AKI model,and glutamine metabolism regulated ferroptosis in SA-AKI through xCT/GSH/GPx4.Part Ⅲ:The mTOR/ATF4/Nrf2 pathway is involved in the regulation of glutamine metabolism on ferroptosis in renal tubules.Objective:To investigate possible molecular mechanisms of glutamine metabolism regulating ferroptosis of renal tubules in SA-AKI.Methods:TKPTS cells were divided into control group,LPS stimulation group,glutamine-deprived group and glutamine deprivation plus LPS stimulation group.The expression of mTOR/ATF 4/Nrf2 was detected by immunoblotting and immunofluorescence.Rapamycin was then used to inhibit mTOR activity and siRNA to silence Atf4 or Nrf2 in glutamine-deprived group.Ferroptosis was dected after LPS stimulation.Results:Compared with LPS-stimulated group,mTOR was activated and the expression of ATF4/Nrf2 was up-regulated and they were transferred to the nucleus in the glutamine-deprived group.After inhibiting mTOR by rapamycin and then stimulating with LPS,the expression of ATF4 and Nrf2 was down-regulated and ferroptosis was increased.When Atf4 was silenced in the glutamin-deprived LPS group,mTOR was activated and Nrf2 expression was down-regulated,and ferroptosis increased.When Nrf2 was silenced in the glutamin-deprived LPS group,mTOR was activated,ATF4 expression didn’t changed,and ferroptosis increased.Conclusions:In LPS model,inhibition of glutamine metabolism activated the mTOR pathway,and mTOR/ATF4 stabilized the xCT/GSH/GPx4 antioxidant system in renal tubular cells by regulating Nrf2,thus alleviating ferroptosis in TKPTS.Part Ⅳ:Inhibition of glutamine metabolism in renal tubular epithelial cells improves sepsis-associated acute kidney injury by reducing ferroptosisObjective:To explore the therapeutic potential of inhibiting glutamine metabolism in SA-AKI.Methods:The cre-loxp system was used to construct tubular-specific Glsf/f mice.SA-AKI model was established via cecal ligation and puncture.The renal function and renal pathological changes of mice were detected.The morphological changes of mitochondria in cortical renal tubules of mice were observed by transmission electron microscopy.The postoperative survival of mice was recorded,and the expression of mTOR/ATF4/Nrf2 was detected by immunoblotting and immunofluorescence.Results:Compared with the SA-AKI group without Gls knockout,renal injury was alleviated and renal ferroptosis reduced in the knockout group.In addition,silencing Gls improved SA-AKI mice survival.mTOR was activated and ATF4/Nrf2 expression was increased in the tubular-specific Glsf/f mice.Conclusions:Inhibition of renal tubular glutamine metabolism can improve the outcome and renal function of SA-AKI mice by inhibiting ferroptosis.